We have discovered the first nucleoside-analog inhibitor (NAI) that selectively inhibits bacterial RNA polymerase (RNAP): pseudouridimycin (PUM). PUM is produced by Streptomyces sp. NAI38640 and comprises a guanidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 5'-amino-pseudoridine. PUM inhibits bacterial RNAP--but not mammalian RNAP--in vitro, inhibits bacterial growth in culture, and potently clears infection in a mouse model of Group A Streptococcus infection (ED50 = 10 mg/kg). The compound exhibits antibacterial activity against a broad spectrum of drug-sensitive and drug-resistant bacterial pathogens, including drug-sensitive, penicillin-resistant, macrolide-resistant, and multi-drug-resistant Streptococci, drug-sensitive, methicillin-resistant, and multi-drug-resistant Staphylococci, Neisseria sp., Haemophilus sp., and Moraxella sp. The compound exhibits no cross-resistance with rifampin, the RNAP inhibitor in current use in broad-spectrum antibacterial therapy, and exhibits a spontaneous resistance frequency <1/10 that of rifampin. The compound exhibits additive antibacterial activity upon co-administration with rifampin. We have defined the binding site on RNAP for PUM (the i+1 NTP insertion site) and the mechanism of inhibition of RNAP by PUM (competition with UTP for occupancy of the i+1 NTP insertion site). The binding site and mechanism have no overlap with the binding site and mechanism of the RNAP inhibitor rifampin, consistent with the absence of cross-resistance with rifampin. We have determined a crystal structure of RNAP in complex with PUM. The crystal structure suggests specific alterations to the structure of PUM that are expected to increase potency against a broad spectrum of bacterial RNAP, exploiting structural features that are invariant in bacterial RNAP. We have developed procedures for semi-synthesis and total synthesis of PUM analogs. We propose to leverage the mechanistic information, structural information, and synthetic procedures obtained in preliminary work in order to design, synthesize, and evaluate PUM analogs having increased efficacy against a broad spectrum of drug-resistant and drug-resistant bacterial pathogens. Analogs will be evaluated for inhibition of RNAP in vitro, antibacterial activity in culture, cytotoxicity against mammalian cells in culture, resistance properties in culture, and physical properties. Analogs of high promise will be evaluated for antibacterial efficacy in small-animal models of infection, and analogs of highest promise will be evaluated for bioavailability, pharmacokinetics, safety, and ability to scale synthesis.